666
LLOYDL. INGRAHAM
ously extracted with ether. Evaporation of the ether from the first ether extraction gave scopolamine (4.2 g.) as a cloudy viscous oil. Evaporation of the ether from the continuous extraction, after drying over anhydrous magnesium sulfate, gave 3.35 g. of scopine as a clear viscous oil. This is a 96% yield based on unrecovered scopolamine. T h e colorless oil was taken up in excess pentane and treated with 0.1 g. of charcoal. -1fter removal of the charcoal, hexane was added and the solution was concentrated by gentle warming. The solution was cooled t o room temperature and then in the refrigerator overnight. This gave crystalline scopine, m.p. 73-75". One further crystallization from pentane gave colorless needles, m.p. 76'. Solid scopine as a h-ujol mull shoTved the folfowing infrared maxima: 3.04, 7.52, 7.65, 7.80, 8.07, 8.23, 8.33, 8.68, 8.85, 9.28, 9.60, 9.77, 10.05, 10.18, 10.60, 11.50, 11.80, 12.29, 13.70 and 14.11 w . Scopine in aqueous solution formed ii picrate in good yield when treated with a methanolic solution of picric acid. The picrate had 11i.p. 242-243' (identical with the melting point of authentic scopoline picrate). -1mixture melting point with authentic scopoline picrate showed no depression.
[ COSTRIBUTIOK PROM THE \\'ESTEKN
Vol. 79
The infrared spectra of scopine picrate and scopoline picrate were, however, distinctly different, each spectrum containing maxima totally absent in the other. The conversion of scopine picrate to scopoline picrate on heating to the melting point is not surprising in view of the report Ly Moffett and Garrett t h a t scopine methobromide rearranges to scopoline methobromide on heating to the ineltirig point .5 Scopine Methobromide.-A solution of scopine in t i i d i anol was treated with excess methyl broinitic and left ovvrnight at 0'. Scopine methobromide \viis then collected in nearly quantitative yield (m.p. 2944296" dec.). Scopoline (III).-Scopolamine, dissolved in excess 10' potassium hydroxide, gave scopoline as ii clear viscous oil in 985; yield after 17 hours reflux. Scopoline showed the following infrared maximi: 2.97, .3.47, 6.86, 6.98, 7.16, 7.31, 7.54, 7.65, 7.74, 8.04, 8.21, 8.35, 8.56, 8.96, 9.31, 9.49, 9.60, 9.68, 9.82, 9.98, 10.20, 11.08, 11.20, 11.44, 12.00, 12.21, 12.50, 12.95 and 14.30 p . Scopoline picrate, obtained by Iriixirig a n aqueous solution of picric acid a n an aqueous solution of scopoline, had m.p. 242-243" dec.
ITHACA, NEWYORK
RESEARCH BRANCH, .iGRICUL,TURAL DEPARTMENT O F A%GRICULTURE]
UTILIZATIOS
I-stein catalyzed by polyphenol oxidase. Three hydrogen donors, catechol, chlorogenic acid and caffeic acid, were chosen for this study. From the assumption t h a t the rate constants governing the equilibrium between enzyme and oxygen are independent of hydrogen donor structure and the fact that the Michaelis constant for oxygen is highly dependent upon the maximum velocity for three different hydrogen donors, the conclusion is drawn that the oxygen Michaelis constant is not a n equilibrium constant. A study is also reported of the variation of the oxygen Michaelis constant with the hydrogen donor concentration. This effect had been predicted previously from theoretical considerations. The Michaelis constants approach a value of at low values of the hydrogen donor concentration regardless of the hydrogen donor structure. These facts ~ 1 . 5 oxygen 7 ~ are consistent with the ideas t h a t the equilibrium constant between enzyme and oxygen is ~ 1 . 5 oxygen 7 ~ and also t h a t the enzyme combines with oxygen before it does with the hydrogen donor.
In a recent paper1 the kinetics for a two-substrate enzyme-catalyzed system were discussed. This discussion showed theoretically how the Michaelis constant for one substratemay vary with the concentration of the other substrate. Previous variations of Michaelis constants have been reported for glucose oxidase,? lipoxidase3and dextransucrase." The purpose of this paper is to report a study of the variation of the Michaelis constant for oxygen with both structure and concentration of the other substrates, the hydrogen donors, in the polyphenol oxidase catalyzed system and also a discussion of the significance of these variations in understanding the mechanism of the polyphenol oxidase catalyzed aerobic oxidation of catechol. Measurements of Michaelis Constants.-The Michaelis constants for the substrate oxygen were determined from the curvature in the plots of the consumption of oxygen v s . time as measured by means of a rotating polarized e l e ~ t r o d e . ~The (1) L. 1,. I n g r a h a m a n d I(. Makower, J . P h y s . Chcin., 68, 26ti (1954); see also J . 2.Hearon, Physiol. Revs., 32, 499 (1952). ( 2 ) H. Laser, Proc. R o y . S O L .(London), 140B,230 (1962). ( 3 ) A. L. T a p p e l , 1'. D. Boyer a n d W. 0 . Lundberg. .7. Bioi. Chcm., 199, 267 (1952). (4) H. M. Tsuchiya and C . S.Stringer, SOC.Am. B a c t . Meeting, New York, X . Y., 1053. ( 5 ) L. 1.. I n n r a h a m , riiinl. Lheii~.,28, 1177 (lY5tij.
decrease in rate of oxygen consumption with tiine may be the result of either or both the diminution of the oxygen supply in solution or a decrease in catalytic activity of the enzyme from reactioninactivation.6,' The hydrogen donor is kept a t a constant concentration by the excess of ascorbic acid in solution. One may calculate when reactioninactivation is important in affecting the curvature as follows : The rate of consumption of oxygen has a Michaelis dependence on oxygen concentration8 I
where k o is the rate constant for oxidation, hyXn is the Michaelis constant and (E) is the enzyme concentration. In addition, the rate of disappearance of 11
has a similar dependence on oxygen concentration.' X combination of equations I and I1 gives k~ d(OJ
kod(E)
I11
( t i ) W, 11. Miller and C . R . l ) , i w s , 1 1 , 'l'ms J O I I K N . X I . 63, 3 3 7 6 (1941). (7) I,. I,. I n g r a h a m , J . Corse and B. 3lakower, ibzii., 74, 2623 (1952). (8) L . L Ingraham zbid 77 2875 ( l Y j 5 ) .
OXYGENMICHAELIS CONSTANT I N OXIDASE-CATALYZED SYSTEMS
Feb. 5, 1957
Equation 111 may be integrated and the value of (E) substituted in equation I. The resulting equation may be integrated t o give equation IV. a (a - s o - K ) In (e VrJ ( a - So)
-
+ s - Sa) + (a)
In the above equation, the ultimate amount of ascorbic acid oxidized a = koEo,/kI and the initial velocity, VO = koEo. The symbols Eo and So denote initial enzyme and oxygen concentrations, respectively, and S denotes the oxygen concentration a t time, t. When So K
